Toll-like receptors (TLRs) regulate activation of the innate immune response and influence the formation of adaptive immunity by detecting and initiating signal transduction cascades in response to bacterial, viral, parasitic, and in some cases host-derived ligands (Lancaster et al., J. Physiol. 563:945-955, 2005). Members of the TLR family TLR1, TLR2, TLR4 and TLR6 are located on the plasma membrane and activate downstream signaling pathways in response to ligands including protein or lipid components of bacteria and fungi. TLR3, TLR7 and TLR9 are preferentially localized intracellularly, and respond to dsRNA, ssRNA and unmethylated CpG DNA, respectively.
TLRs signal through adaptor molecules myeloid differentiation factor 88 (MyD88), Toll/IL-1 receptor domain containing adaptor inducing interferon-beta (TRIF) and TRIF-related adaptor molecule (TRAM), initiating signaling pathways involving JNK/p38 kinase, interferon-regulatory factors (IFN) IFN-3, IFN-5 and IFN-7, and NF-kB, leading to the production of pro-inflammatory cytokines (Romagne, Drug Discov. Today 12:80-87, 2007). TLR3 regions critical for receptor signaling have been identified. Mutations in residues involved in protein glycosylation, disulfide bond formation, loop 2 and leucine rich repeat (LRR) sequences result in signaling deficient TLR3 (Ranjith-Kumar et al., J. Biol. Chem. 282:7668-7678, 2007; Ranjith-Kumar et al., J. Biol. Chem. 282:17696-17705, 2007; Sun et al., J. Biol. Chem. 281:11144-11151, 2006; Takada et al, Mol. Immunol. 44:3633-3640, 2007). Crystal structure of a complex between two murine TLR3 extracellular domains and TLR3 ligand dsRNA further revealed ligand binding amino acids and regions critical for proper folding and dimerization of TLR3 (Liu et al., Science 320:379-81, 2008). TLR3 may also be regulated via alternative splicing. A soluble form of TLR3 was cloned in chicken (Yilmaz et al., Immunogenetics 56:743-53, 2005), and a human TLR3 mRNA encoding a splice variant with alternative splicing of TLR3 exon 4 resulting in 192 bp in frame deletion has been identified (Yang et al., Immunogenetics 56:743-53, 2005). The functional significance of the TLR3 variants described is unknown.
Dysregulation of TLR signaling is believed to cause a multitude of problems, and therapeutic strategies are in development towards this axis (Hoffman et al., Nat. Rev. Drug Discov. 4:879-880, 2005; Rezaei, Int. Immunopharmacol. 6:863-869, 2006; Wickelgren, Science 312:184-187, 2006). For example, antagonists of TLR4 and TLRs 7 and 9 are in clinical development for severe sepsis and lupus, respectively (Kanzler et al., Nat. Med. 13:552-559, 2007).
TLR3 signaling is activated by dsRNA, mRNA or RNA released from necrotic cells upon inflammation or virus infection. TLR3 activation results in induced secretion of interferons and pro-inflammatory cytokines, which have been associated with pathogen infections, and shown to contribute to a spectrum of inflammatory, immune-mediated and autoimmune diseases, for example, asthma, chronic obstructive pulmonary disease, psoriasis, septic shock, rheumatoid arthritis, inflammatory bowel disease such as Crohn's disease and ulcerative colitis, and type I diabetes (Tabeta et al., Proc. Natl. Acad. Sci. 101:3516-3521, 2004; Underhill, Curr. Opin. Immunol. 16:483-487, 2004; Gaspari, J. Am. Acad. Dermatol. 54:S67-80, 2006; Van Amersfoort et al., Clin. Microbiol. Rev. 16:379-414, 2003; Miossec et al., Curr. Opin. Rheumatol. 16:218-222, 2004; Ogata and Hibi, Curr. Pharm. Res. 9:1107-1113, 2003; Takeda and Akira, J. Derm. Sci. 34:73-82, 2004; Doqusan et al., Diabetes 57:1236-1245, 2008).
TLR3 expression has been shown to correlate with inflammatory responses associated with pathological conditions such as primary biliary cirrhosis of liver tissues (Takii et al., Lab Invest. 85:908-920, 2005). Furthermore, TLR3 was found overexpressed in joints of patients with rheumatoid arthritis (Ospelt et al., Arthritis Rheum. 58:3684-92, 2008). TLR3 plays a key role in the immune response upon virus infection. For example, TLR3 deficient animals exhibit a survival advantage over wild type animals upon influenza A virus infection, with the enhancement of survival correlating with reduced levels of pro-inflammatory mediators (Le Goffic et al., PloS Pathog. 2:E53, 2006). TLR3 deficient animals are also protected from rotavirus infection-induced mucosal epithelial breakdown (Zhou et al. J. Immunology 178:4548-4556, 2007). In humans, a dominant-negative TLR3 allele has been associated with increased susceptibility to Herpes Simplex encephalitis upon primary infection with HSV-1 (Zheng et al., Science 317:1522-7 2007).
In necrotic conditions, the release of intracellular content including endogenous mRNA triggers secretion of cytokines, chemokines and other factors that induce local inflammation, facilitate clearance of dead cell remnants and repair the damage. Necrosis often perpetuates inflammatory processes, contributing to chronic or exaggerated inflammation (Bergsbaken et al., Nature Reviews 7:99-109, 2009). Activation of TLR3 at the site of necrosis may contribute to these aberrant inflammatory processes and generate a further pro-inflammatory positive feedback loop via the TLR3 ligands released. Down-modulation of TLR3 activation may also represent a novel treatment strategy for oncologic indications including renal cell carcinomas and head and neck squamous cell carcinomas (Morikawa et al., Clin. Cancer Res. 13:5703-5709, 2007; Pries et al., Int. J. Mol. Med. 21: 209-15, 2008). Also, a previously characterized TLR3L423F allele resulting in reduced TLR3 activity was associated with protection against advanced “dry” age-related macular degeneration (Yang et al., N. Engl. J. Med. 359:1456-63, 2008), indicating that TLR3 antagonist agents may be beneficial in this disease.
Pathologies associated with inflammatory conditions and others, such as those associated with infections, have significant health and economic impacts. Yet, despite advances in many areas of medicine, comparatively few treatment options and therapies are available for many of these conditions.
Thus, a need exists to suppress TLR3 activity to treat TLR3-associated conditions.